More than 80 percent of global transmission of HIV infection occurs via mucosal routes. The ability of vaccines to induce mucosal immunity may be required for protection against HIV infection and the immunodeficiency syndrome that emerges after infection. Various AIDS vaccine candidates are currently under development, each with potential advantages and disadvantages. DNA vaccines have features that make them attractive vaccine candidates for HIV and the combination of DNA vaccination plus recombinant vaccinia is one the favored approaches in AIDS vaccine development. We have established a vaccination regiment that consistently stimulates mucosal responses with an SIV DNA vaccine, which consist of a single plasmid carrying an SIV proviral genome that produces noninfectious particles. We have appropriate tools to measure SIV-specific systemic responses. As a consequence , we believe that we are uniquely placed to establish the effectiveness of mucosal DNA vaccination and how stimulation of virus-specific mucosal responses can affect the efficacy of a HIV/SIV vaccine. This proposal is designed to extend our previous studies by investigating approaches to maximize immune responses to DNA vaccination in primates using a SHIV contrast based on our SIV proviral genome vaccine. Toward this goad we propose to evaluate whether: 1. the immunostimulatory properties of a DNA vaccine can be amplified and made consistent in an outbred population by the addition of genes expressing cytokines and heat shock proteins; 2. an immunization regiment for the SHIV vaccine that involves different mucosal and systemic routes can provide consistently high levels of systemic and mucosal responses within the same animal; 3. the immune responses induced by the different DNA vaccination regimens contribute to protection of macaques from the establishment of persistent SHIV infection and/or simian AIDS after mucosal SIV challenge. The results of this investigation should be important not only for AIDS vaccine development but also for the development of DNA vaccines for other mucosal pathogens.